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USDA/APHIS Petition 95-352-01P for Determination of Nonregulated Status for CZW-3 Squash

Environmental Assessment and

Finding of No Significant Impact

June 1996

The Animal and Plant Health Inspection Service (APHIS) of the U. S. Department of Agriculture has conducted an environmental assessment before issuing a determination of nonregulated status for a genetically engineered squash called CZW-3 Squash. APHIS received a petition from the Asgrow Seed Company regarding the status of the CZW-3 Squash as a regulated article under APHIS regulations at 7 CFR Part 340. APHIS has conducted an extensive review of the petition and supporting documentation, and other relevant scientific information. Based upon the analysis documented in this environmental assessment, APHIS has reached a finding of no significant impact on the environment from its determination that virus resistant CZW-3 Squash shall no longer be a regulated article.

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John H. Payne, Ph.D.

Acting Director

Biotechnology, Biologics, and Environmental Protection

Animal and Plant Health Inspection Service

U.S. Department of Agriculture

Date:

TABLE OF CONTENTS

I. SUMMARY3

II. BACKGROUND4

III. PURPOSE AND NEED6

IV. ALTERNATIVES6

V. AFFECTED ENVIRONMENT AND POTENTIAL ENVIRONMENTAL IMPACTS6

VI. CONCLUSIONS10

VII. LITERATURE CITED12

VIII. PREPARERS AND REVIEWERS12

IX. AGENCY CONTACT13

I. SUMMARY

The Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) has prepared an Environmental Assessment (EA) prior to making a determination on the regulated status of a genetically engineered, virus resistant line of yellow crookneck squash (Cucurbita pepo subsp. ovifera var. ovifera) designated CZW-3 squash. The developer of CZW-3 squash, the Asgrow Seed Company, petitioned APHIS requesting the determination on the regulated status of CZW-3, a plant that has been a regulated article under APHIS regulations. Under APHIS regulations importation, interstate movements and field tests of CZW-3 squash have required permits issued by APHIS. Asgrow has petitioned APHIS for a determination that CZW-3 squash does not present a plant pest risk and should therefore no longer be a regulated article under the APHIS regulations found at 7 CFR Part 340.

The CZW-3 squash has been developed to resist infection by three plant viruses that infect squashes. The genes conferring viral resistance in CZW-3 were introduced via genetic engineering techniques. Those techniques enabled the developer to introduce into yellow crookneck squash viral coat protein genes from cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV), and watermelon mosaic virus (WMV2). Incorporation of these coat protein genes into the squash plants does not cause plant disease, but rather enables CZW-3 squash plants to resist infection by CMV, ZYMV, and WMV2. The genes were introduced into CZW-3 via a well-characterized procedure that utilizes the bacterium Agrobacterium tumefaciens to introduce genes into plant genomes.

From 1993 through 1996, APHIS has issued 10 permits to Asgrow to conduct field tests with CZW-3 squash. APHIS prepared EAs prior to granting the field test permits. Previous EAs addressed issues pertinent to plant pest risk issues relative to field tests conducted under physical and reproductive confinement, but they did not address several issues relevant to the unconfined growth of CZW-3 squash. With respect to the unconfined growth of CZW-3 squash, APHIS has reached the following conclusions:

1. CZW-3 squash exhibits no plant pathogenic properties. Although plant pathogenic organisms were used in the development of CZW-3 squash, these squash plants are not infected, nor can they incite disease in other plants.

2. CZW-3 squash is no more likely to become a weed than a virus-resistant squash plant developed by traditional breeding techniques. Squash is not considered to be a weed pest, and there is no reason to believe that the ability of CZW-3 squash to resist infection by CMV, ZYMV, and WMV2 will lead to this squash becoming a weed pest. The introduction of traditionally-bred, improved squash varieties has not resulted in squashes that are considered weeds.

3. CZW-3 squash will not increase the weediness potential for any other cultivated plant or native wild species with which it can interbreed. As with other cultivated squashes, it will be possible for the pollen of CZW-3 squash to pollinate free-living Cucurbita pepo (FLCP) plants, the so-called wild relatives of cultivated squash. Although genes can move freely via pollen from CZW-3 squashes to FLCP plants, there is no indication that such cross-pollination will result in hybrid offspring that present any significant increase in their weediness.

4. CZW-3 squash will not cause damage to processed agricultural commodities.

5. CZW-3 will not increase the likelihood of the emergence of new plant viruses. APHIS has carefully considered the biology and epidemiology of the plant viruses that infect squash, and APHIS has determined that the unconfined cultivation of CZW-3 squash would be no different than traditionally bred, virus resistant squash cultivars with respect to the appearance of new plant viruses.

6. CZW-3 squash will not harm threatened or endangered species or other organisms, such as bees, which are beneficial to agriculture.

APHIS has also concluded that there is no reason to believe that new progeny CZW-3 squash varieties bred from these lines should not exhibit new plant pest properties, i.e., properties substantially different from any observed for the CZW-3 squash lines already field tested, or those observed for squashes in traditional breeding programs.

Therefore, after review of the available evidence, APHIS concludes that CZW-3 squash will be just as safe to grow as virus resistant squash cultivars developed through traditional breeding practices. The cultivation of CZW-3 squash should present environmental impacts that are no different from the impacts associated with traditionally-bred squash varieties that are not subject to regulation under 7 CFR Part 340 before they enter agriculture. Based upon the analysis documented in this EA, APHIS has reached a finding of no significant impact on the environment from its determination that the CZW-3 squash will no longer be considered a regulated article under the regulations in 7 CFR Part 340.

II. BACKGROUND

A. Development of CZW-3 Squash

The CZW-3 squash has been developed to resist infection by three plant viruses that commonly infect squashes. The genes conferring viral resistance in CZW-3 were introduced via recombinant DNA (genetic engineering) techniques rather than conventional breeding techniques. The recombinant techniques enabled the developer to introduce three viral coat protein genes from plant viruses into the yellow crookneck squash. The genes were obtained from cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), watermelon mosaic potyvirus (WMV2). The latter two viruses are members of the potyvirus group of plant viruses while the former is a member of the cucumovirus group. Incorporation of these genes into the squash to yield the CZW-3 plants does not cause plant disease, but rather enables the plants to resist infection by CMV, ZYMV, and WMV2. The genes were introduced into CZW-3 via a procedure mediated by a strain of the plant pathogenic bacterium Agrobacterium tumefaciens, which has been disarmed so that it is no longer pathogenic to plants. This procedure is well characterized and has been used widely for over a decade as a means of introducing various genes of interest directly into plant genomes.

CZW-3 squash lines have been evaluated extensively in laboratory, greenhouse, and field experiments to confirm that they exhibit the desired agronomic characteristics and that they do not present a plant pest risk. Through the end of 1996, APHIS has issued 10 permits for field tests of CZW-3 squash. The field tests of CZW-3 have been conducted in controlled agricultural settings, under permit conditions that have stipulated physical and reproductive confinement of the CZW-3 plants.

B. APHIS Regulatory Authority

APHIS regulations at 7 CFR Part 340, which were promulgated pursuant to authority granted by the Federal Plant Pest Act (7 U.S.C. 150aa-150jj), as amended, and the Plant Quarantine Act (7 U.S.C. 151-164a, 166-167), as amended, regulate the introduction (importation, interstate movement, or release into the environment) of certain genetically engineered organisms and products. A genetically engineered organism is considered a regulated article if the donor organism, recipient organism, vector or vector agent used in engineering the organism belongs to one of the taxa listed in the regulation and is also a plant pest, or if there is reason to believe that it is a plant pest. The transgenic squash plants described in the Asgrow petition have been considered regulated articles because noncoding DNA regulatory sequences and portions of the plasmid vector were derived from plant pathogens.

An organism is not subject to the regulatory requirements of 7 Part 340 when it is demonstrated not to present a plant pest risk. Section 340.6 of the regulations, entitled "Petition Process for Determination of Nonregulated Status," provides that a person may petition the agency to evaluate submitted data and determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If the agency determines that the regulated article is unlikely to pose a greater plant pest risk than the unmodified organism, APHIS can grant the petition in whole or in part. As a consequence of determining nonregulated status, APHIS permits are no longer required for field testing, importation, or interstate movement of that article or its progeny.

III. PURPOSE AND NEED

APHIS has prepared this EA prior to making a determination on the status of CZW-3 squash as a regulated article under APHIS regulations. The developer of CZW-3 squash, Asgrow, submitted a petition to USDA/APHIS requesting that APHIS make a determination that CZW-3 squash shall no longer be considered a regulated article under CFR Part 340. This EA was prepared in compliance with: (1) the National Environmental Policy Act of 1969 (NEPA)(42 U.S.C. 4321 et seq.), (2) Regulations of the Council on Environmental Quality for implementing the procedural provisions of NEPA (40 CFR parts 1500-1508), (3) USDA regulations implementing NEPA (7 CFR part 1b), and (4) APHIS' NEPA Implementing Procedures (7 CFR part 372; 60 FR 6000-6005, February 1, 1995).

IV. ALTERNATIVES

A. No Action

Under the Federal "no action" alternative, APHIS would not come to a determination that CZW-3 squash is no longer a regulated article under the regulations at 7 CFR Part 340. Permits from APHIS would still be required for introductions of CZW-3 squash. APHIS might choose this alternative if there were insufficient evidence to predict the lack of plant pest risk from unconfined cultivation of CZW-3 squash.

B. Determination That CZW-3 Squash is No Longer a Regulated Article

Under the Federal action to render a determination that CZW-3 squash is no longer a regulated article under the regulations at 7 CFR Part 340, CZW-3 squash would be subject to the same regulatory oversight as cultivars that result from traditional breeding practices. As such, permits from APHIS would no longer be required for introductions of CZW-3 squash or its progeny.

V. AFFECTED ENVIRONMENT AND POTENTIAL ENVIRONMENTAL IMPACTS

This EA addresses potential environmental impacts from a determination that CZW-3 squash would no longer be considered a regulated article under APHIS regulations at 7 CFR Part 340. Previous EAs prepared by APHIS in conjunction with the issuance of permits for field tests of CZW-3 have addressed various attributes of this squash line. This EA discusses the genetic modification of this squash line, the resultant phenotype, and the potential environmental impacts that might be associated with the unconfined cultivation of CZW-3 squash.

Additional technical information is included in the determination document appended to this EA, and incorporated by reference. This includes detailed discussions of the biology of cucurbits, the genetic components used in the development of CZW-3 squash, and another analysis that supports APHIS' conclusion that CZW-3 squash has no potential to pose plant pest risks.

A. Potential for the Appearance of New Plant Viruses

As mentioned above, CZW-3 squash was developed by engineering the viral coat protein genes of CMV, ZYMV, and WMV2 into a cultivar of yellow crookneck squash, a plant which is frequently infected by these and other plant viruses. As part of its analysis, APHIS evaluated whether the expression of these viral genes in CZW-3 squash might present some unusual circumstances that could lead to the appearance of new plant viruses.

In the course of the infection of a plant cell by more than a single type of virus, it is possible for some of the constituents of the viruses to become mismatched. Such occurrences can lead to recombination of the nucleic acid genome or a mixture of the protein subunits (called - transcapsidation), which comprise the coat of the virus particle. It is theoretically possible for new plant viruses to arise in the CZW-3 squash through the recombination or transencapsidation, and APHIS considered this issue carefully in making its determination. A technical discussion of this issue is found in the Determination document appended to this EA. After careful consideration of the physical and biological properties of CMV, ZYMV, and WMV2, APHIS concluded that it is unlikely that new viruses will appear as a consequence of the widespread cultivation CZW-3 squash. Current control measures are adequate to control any potential new virus.

B. Potential Impacts Based on Increased Weediness of CZW-3 Squash Relative to Traditionally Bred Squash

APHIS evaluated whether the CZW-3 squash itself is likely to present a plant pest risk as a weed. The parent plant in this petition, yellow crookneck squash, is an agricultural crop plant that exhibits no appreciable weedy characteristics. None of the standard texts and lists of weeds indicate that squash is regarded as a weed (Holm et al., 1979; Muenscher, 1980; Reed, 1970; Weed Science Society of America, 1992).

The relevant introduced trait, resistance to infection by CMV, ZYMV, and WMV2, is unlikely to make the CZW-3 squash into a weed. Resistance or tolerance to pests is commonly bred into agricultural crops, including squash. Despite this, improved squash cultivars have not become weeds. Likewise, there is no indication that resistance to ZYMV and WMV2 will result in CZW-3 squash becoming a weed (see the Determination).

No other attributes of CZW-3 squash suggest that it is any more "weedy" than squash cultivars that are the result of traditional breeding. The CZW-3 squash has retained the agronomic characteristics of the parental crookneck squash.

C. Potential Impacts on the Free-Living Relatives of Squash Arising From Pollination by CZW-3 Squash

APHIS evaluated two potential impacts that CZW-3 squash might have on the free-living relatives of squash. First, that the traits from CZW-3 squash might cause the free-living relatives to become "weedier." Second, that the pollination of free-living populations of squashes would cause population changes that would lead to reduced genetic diversity.

Successful transmission of genetic material from CZW-3 squash via pollen is possible to a limited number of squash relatives (Wilson, 1993). In the United States, the squash relatives that might be successfully pollinated by CZW-3 squash and produce offspring are Cucurbita pepo subsp. texana and C. pepo subsp. ozarkana, plants referred to here and in the Determination as free-living Cucurbita pepo (FLCP). In the past, these FLCP plants have been cited as weeds in soybean and cotton fields (Weed Science Society of America, 1992), but the agricultural significance appears to be minimal with the advent of effective control practices over the past decade (see Determination).

It is unlikely that offspring arising from natural crosses of FLCP and CZW-3 will pose a weed problem. Current agricultural production of squash in the United States occurs near the habitats where FLCP plants are found in the Southeastern States. This proximity is sufficient for the pollination of FLCP by squash cultivars, but there has been no apparent emergence of weedy hybrid progeny (see the Determination). This has not occurred even as plant breeders continue to develop cultivated squash varieties with enhanced pest resistance qualities. Typically, breeders seek out free-living (wild) relatives as a source of pest resistance traits to cross into a cultivated crop.

In addition, it is clear that any progeny of cultivated and free-living squashes will receive a set of genetic material from each of the parents. In this case, the cultivated squash parent contributes genetic material responsible for ensuring a plant that produces tender-skinned fruits that have low levels of cucurbitin, a bitter-tasting compound that discourages feeding by herbivores. Invariably, the FLCP is better adapted than commercial squash cultivars to survival in the absence of cultivation. Thus, there has been no report to date of weed problems arising from the possible crosses that might occur between domesticated varieties of squash and their free-living relatives.

Given the available knowledge, it is unlikely that resistance to CMV, ZYMV, and WMV2 infection will confer a selective advantage or be maintained in the FLCP populations. Surveys of natural FLCP populations for the incidence and severity of CMV, ZYMV, and WMV2 infections suggest that resistance to these viruses will confer little, if any, selective advantage, because disease caused by these viruses is apparently not among the factors important to the survival or reproductive success of FLCP (see the Determination).

Based upon our analysis of the biology of cultivated squash and its relatives, APHIS concludes that the environmental impacts of cultivation of CZW-3 squash anywhere in the world will be no different than such impacts attributable to similar varieties produced with traditional breeding techniques. The species Cucurbita pepo is native to the North American continent, with a center of biological diversity in northern Mexico, and a center of diversity (probably secondary, though embracing a greater variety) in the Southeastern United States. Cultivated and noncultivated varieties of C. pepo have coexisted and co-evolved over millennia. Even if CZW-3 squash were to be cultivated in agricultural regions around centers of C. pepo diversity, there is no reason to expect impacts from CZW-3 squash would be significantly different from those arising from the cultivation of any other variety of squash. As discussed above, natural populations of FLCP appear to be largely free of infection by ZYMV and WMV2. It therefore appears that resistance to ZYMV and WMV2 should not provide any selective advantage. Without a selective advantage, this trait is unlikely to persist in the gene pool of FLCP.

There is already considerable cultivation of traditional squash varieties throughout the centers of diversity for C. pepo, including virus resistant varieties. Conventionally-bred virus resistant cucurbits that are available for use include: Harris Moran's zucchini squash that is resistant to ZYMV and WMV-2 and zucchini squash (specific virus not described but possibly CMV) and CMV-resistant marrow squash that are available commercially from Thompson and Morgan, Inc. of Jackson, New Jersey. The impact of cultivation of CZW-3 squash on the genetic diversity of FLCP populations is likely to be comparable to that from nontransgenic varieties.

We note also that any international traffic in CZW-3 squash would be fully subject to national and regional phytosanitary standards promulgated under the International Plant Protection Convention (IPPC). The IPPC has set a standard for the reciprocal acceptance of phytosanitary certification among the nations that have signed or acceded to the Convention (98 countries as of December 1992). The treaty, now administered by a Secretariat housed with the United Nations Food and Agriculture Organization in Rome, came into force on April 3, 1952. It establishes standards to facilitate the safe movement of plant materials across international boundaries. Plant biotechnology products are fully subject to national legislation and regulations, or regional standards and guidelines promulgated under the IPPC. The vast majority of IPPC signatories have promulgated, and are now administering, such legislation or guidelines, including Mexico, which has in place a regulatory process that would require a full evaluation of the CZW-3 squash before it could be introduced into their environment. Our decision in no way prejudices regulatory action in Mexico or any other country. The IPPC has also led to the creation of Regional Plant Protection Organizations such as the North American Plant Protection Organization (NAPPO). Our trading partners will be kept informed of our regulatory decisions through NAPPO, and other fora. In addition to the assurance provided by the analysis leading APHIS to a finding of no significant impact for the introduction of this squash variety, it should be noted that all the considerable, existing national and international regulatory authorities and phytosanitary regimes that currently apply to introductions of new squash varieties internationally apply equally to those covered by this analysis.

D. Potential Impact on Nontarget Organisms, Including Beneficial Organisms Such as Bees and Earthworms

Consistent with its statutory authority, APHIS evaluated whether CZW-3 squash might indirectly harm plants or plant products (such as some agricultural commodities). APHIS considered the potential impact that CZW-3 might exert indirectly on organisms that are recognized as beneficial to agriculture. APHIS concludes that there is no reason to believe that the unconfined growth of CZW-3 squash will pose any deleterious effects or significant impacts on nontarget organisms, including beneficial organisms. The coat proteins expressed in CZW-3 squash are not known to have any toxic properties. In fact, these viral coat proteins are routinely ingested by virtually all animals, including humans, when squash is consumed. Naturally occurring infections of susceptible squash varieties result in concentrations of coat proteins far higher than those that occur in the tissues of the CZW-3 squash (see the Determination).

APHIS believes that CZW-3 squash will have no deleterious effects on organisms recognized as beneficial to agriculture (e.g., earthworms, honeybees). In addition, there is no reason to believe that the presence of CZW-3 squash would have any adverse effect on other organisms, including any species recognized as threatened or endangered in the United States. The release of CZW-3 squash from regulation should have no adverse impact on agricultural commodities.

VI. CONCLUSIONS

APHIS has evaluated information from the scientific literature as well as data submitted by Asgrow that characterize the CZW-3 squash. After careful analysis, APHIS has identified no significant impact to the environment from issuance of a determination that CZW-3 squash would no longer be a regulated article under APHIS regulations at 7 CFR Part 340.

APHIS has considered the foreseeable consequences of removing CZW-3 from its regulation and reached the following conclusions:

3. CZW-3 squash is unlikely to increase the weediness potential for any other cultivated plant or native wild species with which it can interbreed. As with other cultivated squashes, it will be possible for the pollen of CZW-3 squash to pollinate free-living Cucurbita pepo (FLCP) plants, the so-called "wild" relatives of cultivated squash. Although genes can move freely via pollen from CZW-3 squashes to FLCP plants, there is no indication that such cross-pollination will result in hybrid offspring that present any significant increase in their weediness.

4. CZW-3 squash will not cause damage to processed agricultural commodities.

6. CZW-3 squash will not harm other organisms, such as bees, which are beneficial to agriculture.

APHIS has also concluded that new progeny CZW-3 squash varieties bred from these lines should not exhibit new plant pest properties, i.e., properties substantially different from any observed for the CZW-3 squash lines already field tested, or those observed for squashes in traditional breeding programs.

Therefore, after review of the available evidence, APHIS concludes that CZW-3 squash will be just as safe to grow as virus resistant squash cultivars developed through traditional breeding practices. The cultivation of CZW-3 squash should present environmental impacts that are no different from the impacts associated with traditionally-bred squash varieties that are not subject to regulation under 7 CFR Part 340 before they enter agriculture. Based upon the analysis documented in this Environmental Assessment, APHIS has reached a finding of no significant impact on the environment from its determination that the CZW-3 squash will no longer be a regulated article under the regulations in 7 CFR Part 340.

VII. LITERATURE CITED

Holm, L., Pancho, J.V., Herbarger, J.P., and Plucknett, D.L. 1979. A Geographical Atlas of World Weeds. John Wiley and Sons, New York. 391 pp.

Muenscher, W.C. 1980. Weeds (2d ed.). Cornell University Press, Ithaca and London. 586 pp.

Reed, C. F. 1970. Selected Weeds of the United States. Agriculture Handbook No. 366. Agricultural Research Service, U.S. Department of Agriculture, Washington, DC. 463 pp.

Weed Science Society of America. 1992. Composite List of Weeds. WSSA. Champaign, Illinois.

Wilson, H. 1993. Free-living Cucurbita pepo in the United States. Viral resistance, gene flow, and risk assessment. Report to USDA Biotechnology, Biologics and Environmental Protection. 24 pp.

VIII. PREPARERS AND REVIEWERS

Biotechnology, Biologics, and Environmental Protection

John Payne, Ph.D., Acting Director

Biotechnology Permits

Arnold Foudin, Ph.D., Deputy Director

Subhash Gupta, Ph.D., Staff Biotechnologist

David S. Heron, Ph.D., Staff Biotechnologist

Susan M. Koehler, Ph.D., Staff Biotechnologist

James Lackey, Ph.D., Biological Safety Officer

Vedpal S. Malik, Ph.D., Staff Biotechnologist

Henry K. Reding, Ph.D., Staff Biotechnologist

Sivramiah Shantharam, Ph.D., Team Leader, Microorganisms Branch

James L. White, Ph.D., Team Leader, Plants Branch ( Preparer)

Biotechnology Coordination and Technical Assistance

Michael A. Lidsky, J.D., LL.M., Deputy Director

L. Val Giddings, Ph.D., Team Leader, Science Policy Coordination Branch

Shirley P. Ingebritsen, M.A., Regulatory Analyst

Michael Schechtman, Ph.D., Team Leader, Domestic Policy Branch

Frank Y. Tang, Ph.D., J.D., Biotechnologist

Environmental Analysis and Documentation

Carl Bausch, J.D., Deputy Director

IX. AGENCY CONTACT

Ms. Kay Peterson, Regulatory Analyst

Biotechnology Permits, Unit 147

4700 River Road

Riverdale, MD 20737-1237

Phone: (301) 734-7601 Fax: (301) 734-8669